Surface plasmon resonance based nanoliter tear osmometer

ABSTRACT

A medical diagnostic method utilizes a surface plasmon resonance apparatus provided with a sensing surface. A tear sample from an eye of a patient is placed into contact with the sensing surface. The surface plasmon resonance apparatus is then operated to determine osmolarity of the tear sample.

FIELD OF THE INVENTION

This invention relates to an apparatus for measuring the osmolarity ofsolution, and is especially useful for measuring human tears. Theapparatus measures osmolarity of an unknown solution whose volume is onthe order of nanoliters, and is useful for detecting the presence anddegree of dry eye syndrome.

BACKGROUND OF THE INVENTION

Dry Eye Syndrome, or Keratoconjunctivitis Sicca (KCS) is one of the mostfrequently established diagnoses in ophthalmology. Current estimateshold that roughly 40-60 million people in the United States exhibit dryeye symptoms. The lack of accurate statistical data about the occurrenceof dry eye is due largely to a lack of state-of-the-art diagnosticequipment. A more disturbing trend, however, is the misdiagnosis of dryeye or its escape from early detection altogether, since symptomaticpatients are not always easily identified.

Pursuing more effective diagnosis will strengthen the paradigm ofophthalmic care available in this country. The pharmaceutical industryrecognizes this. The first prescription pharmaceuticals for treating dryeye are now appearing on the market, with more on the way in the nexttwenty-four months, and yet the methods for diagnosis and monitoringtreatment remain problematic.

There is no ‘gold standard’ test that both diagnoses dry eye andmonitors the effect of its treatment. The popular method is a matrix ofsubjective observation of symptoms and objective tests (such as Schirmertesting, staining techniques and tear break-up time) none of which isspecific to the detection of dry eye or the measurement of its severity.

Considering recent pharmaceutical advancements aimed at treating dryeye, timely and parallel advancements in diagnostic technologies areneeded.

The osmolarity of a tear—the degree of dissolved solids therein—ispopularly accepted by experts in the field as an indicator of thepresence and severity of dry eye. The instrument most commonlyassociated with the measurement of tear osmolarity is the osmometer;however, technical limitations have restricted the use of tearosmometers to primarily research environments.

An osmometer is a device that measures the concentration of dissolvedsolutes in water. Though it is widely used in other fields, osmometersare used in medicine in applications such as determining osmol gap intoxicology and trauma cases, monitoring mannitol treatment infusions,monitoring the absorption in Glycine ingestion with irrigation fluids insurgical procedures, among others.

Despite the suitability of this technology for measuring tearosmolarity, the current devices present certain limitations that preventtheir widespread use in a clinical environment. The most prevalentproblem has to do with sample size.

Nearly all commercially available osmometers are designed (and perhapstechnologically limited) to measure milliliter-size samples. Tearsamples extracted from patients tend to be in the nanoliter volumes andfurther complicating matters, dry eye patients generally have less tearsand make handling samples even more difficult. The only osmometerdesigned to measure nanoliter sample sizes, is no longer availablecommercially and is too cumbersome for practical use in a clinicalenvironment. The result is that practicing ophthalmologists are leftwith a haphazard methodology and inadequate tools to accurately detectthis prevalent condition.

Dry Eye Syndrome

Dry Eye Syndrome is a complex group of diseases characterized by adecreased production of one or more of the three components of the tearfilm: the lipid layer, the aqueous layer, and the mucin layer. Adeficiency in one of the tear film components may lead to loss of thetear film stability. Normal sight relies on a moist ocular surface andrequires a sufficient quality of tears, normal composition of the tearfilm, regular blinking and normal lid closure as prerequisites. If leftuntreated, dry eye syndrome can cause progressive pathological changesin the conjunctival and corneal epithelium, discomfort, cornealulceration's and even ultimately lead to blindness.

Standard treatment has been tear replacement therapy, which attempts toeither mimic the human tear film or present a more sophisticatedhypo-osmolar version of the tear film. Unfortunately, as dry eyesyndrome progresses beyond the mild stage, this common therapy becomesless effective. Further, these treatments do not address the etiology ofdry eye.

The precise mechanisms that give rise to dry eye are currently unknownand have been under many debates over the years. Several differentmechanisms have been proposed as a possible etiology of dry eye over therecent years, with a general ideology that it is usually caused by aproblem with the quality of the tear film that lubricates the ocularsurface. More recent research has proposed that dry eye may be a resultof a decrease in hormonal status with aging (being more prominent inpostmenopausal women), or have an immune basis and acquired inflammatorycondition of the ocular surface. Other causes of dry eye symptoms canoccur from certain medications (i.e. antihistamines, beta-blockers),associations with certain systemic inflammatory diseases (i.e.rheumatoid arthritis), mechanical causes (i.e. incomplete closure ofeyelids), infectious causes (i.e. viral infections) and certainneurological causes (i.e. LASIK procedures). Despite the recent gains inknowledge of possible pathogenic factors of dry eye, there has been alack of consensus as to the appropriate diagnostic criteria, thespecific aims of objective diagnostic testing, the role subjectivesymptom's play in diagnosis and the interpretation of results.

The symptoms of dry eye vary considerably from one individual toanother. Most patients complain of a foreign body sensation, burning andgeneral ocular discomfort. The discomfort is typically described as ascratchy, dry, sore, gritty, smarting or burning feeling. Discomfort isthe hallmark of dry eye because the cornea is richly supplied withsensory nerve fibers.

Despite its high prevalence, dry eye is not always easy to diagnose. Thevast majority of patients have symptoms that are mild to moderate inseverity. Although these patients are genuinely suffering discomfort,objective signs of dry eye may be missed, and without proper diagnosis,patients may not receive the attention and treatment that this conditionwarrants. The signs and symptoms of dry eye can be misinterpreted asevidence of other conditions such as infectious, allergic, or irritativeconjunctivitis. Given these complications in diagnosis, it is estimatedthat diagnosis rate of dry eye is 20%.

Several drug companies and research sites are formulating drugs tocombat and relieve the symptoms of dry eye, many in FDA Phase III trialsof their version of treatment. At the date of this writing, the firstprescription product for treatment of dry eye is available on themarket, followed by others slated for release in as early as 2004 andtwo others in 2005 and 2006. With no easy way to objectively measure theoccurrence of dry eye, the physicians will be left to subjectivedispensing of the drug, or either late or misdiagnosis.

Current Objective Diagnostic Methods

Diagnosis of dry eye typically begins with clinical examination. ASchrimer test is usually performed where standardized strips of filterpaper are placed at the junction between the middle and lateral third ofthe lower lid. If after 5 minutes less than 5 millimeters has beenwetted there is reason to believe aqueous tear-deficient is present.Though the test is quick, inexpensive and results are availableimmediately, it provides only a rough estimate and is unreliable inmoderate dry eye.

Dye staining is another method of diagnosing dry eye, with eitherfluoriscein or Rose Bengal, and a trained physician can look forpatterns under slit lamp observation indicating dryness. Another test,tear break-up time, is a measure of the stability of the tear film. Anormal tear film begins to break up after approximately 10 seconds, andaccelerated with patients with dry eye.

The osmometer generally used in measuring tear osmolarity is the CliftonDirect Reading Nanoliter Osmometer (Clifton Technical Physics, Hartford,N.Y.) developed in the 1960's. Although not necessarily originallyintended for use in measuring tears, it is one of the few instrumentscapable of measuring nanoliter volumes of solution and has found its wayinto ophthalmology.

The Clifton Osmometer was produced in limited quantities over the years,and is not routinely used outside a research laboratory. It is based onthe well-known measurement technique called freezing point depression.The Clifton Osmometer measures the osmolarity by measuring the freezingpoint depression of the tears. In freezing point depressionmeasurements, water which normally freezes at 0° C., in presence ofdissolved solutes there occurs a depression in its freezing temperature,the mathematical relationship of which is defined by Raoult's Law.

Though the test can be accurate it requires a very skilled operator tomake the measurement. The test monitors the ‘depression’ by examining afractional volume of a teardrop under a microscope. Due to itslimitations and lack of availability, there appears to be only a fewunits left in the field. Furthermore each measurement can take overfifteen minutes, which coupled with the small sample volumes make theuse of Clifton Osmometer an extremely tedious and inconvenient process.The amount of time required and the operating skill demanded areunacceptable to a busy practice or clinic, even if the units wereavailable.

What is direly needed in the hands of a physician performing routineexaminations is a simple but accurate device that can screen, track, andhelp administer treatment and medications at an early stage.

OBJECTS OF THE INVENTION

It is an object of the invention to provide a means to measure theosmolarity of tears for diagnosis of dry eye syndrome.

A more particular object of the invention is to provide an apparatusthat overcomes at least some of the problems inherent in conventionalosmometers, and in particular when measuring osmolarity of nanolitersize samples.

It is further an object of the invention to provide an apparatus thatcan be used clinically—that is simple to use, cost effective for thephysician and patient, and accurate, thereby overcoming the problemsassociated with tear osmometers which are primarily confined to aresearch setting.

These and other objects of the present invention will be apparent fromthe drawings and description herein.

SUMMARY OF THE INVENTION

The present invention is directed in part to an apparatus that measuresthe osmolarity nanoliter volumes of solution including human tears. Theapparatus includes a Surface Plasmon Resonance Spectroscope to determinethe osmolarity of a human tear sample. More particularly, the apparatusincludes a light source, typically a laser, lenses, mirrors, prisms, athin film sensing surface, and an optical detector. The apparatus alsoincludes a computer programmed to analyze raw data and output osmolarityvalues of the tested solution.

Surface Plasmon Resonance (SPR) is a phenomenon that occurs when lightis incident on a metal dielectric interface at a particular angle, sothat the reflected light is extinguished. At the particular angle ofincident light, the intensity of the reflected light shows acharacteristic curve of diminishing intensity well defined bymathematical equations. The energy of the light that is not reflected,is absorbed by the metallic surface and causing an oscillatory mode(resonance) of the electrons in the metal. The first direct measurementof surface plasmon oscillations was observed in 1959, and they have beenextensively studied in the scientific research community throughout the1970's. Scientists have used the technique for such things as measuringrefractive index, properties of metallic thin films, and only in 1982was it suggested that SPR would be useful as a chemical sensor. Sincethen, SPR has grown to a versatile technique used in a variety ofapplications. These include absorbance studies, bio-kinetics andbio-sensing measurements, bulk liquid measurements, gas detection,refractive index measurements, and thin film characterization. In 1992,SPR systems started to appear on the market commercially as a newmeasurement tool for molecular scale research. One of the major appealsof SPR systems was the inherent sensitivity of the measurement, andsupplemented shortcomings found in other forms of spectroscopicmeasurements.

The essential components of an SPR measurement system are relativelysimple. Consisting mainly of a laser source, simple lenses, a thin-filmsensing surface, and a detector, the system can be made compact withlittle effort. One version of a sensing surface consists of a thin filmof gold, 50 nanometers thick, which is deposited on glass by means ofevaporated thin film technology. The thin gold coating is in practice,difficult to produce without significant experience or by performingiterations to achieve the exact thickness required for the phenomenon tooccur. However, a process is described herein for the production of aproprietary thin film coating that produces a strong SPR signal, isdurable and well suited for measuring tear osmolarity.

A medical diagnostic method comprises, in accordance with the presentinvention, providing a surface plasmon resonance apparatus, providingthe surface plasmon resonance apparatus with a sensing surface, placinga tear sample from an eye of a patient into contact with the sensingsurface, and operating the surface plasmon resonance apparatus todetermine osmolarity of the tear sample.

Te surface plasmon resonance apparatus may take the form of a portableprobe. In that event, the placing of the tear sample comprisesmanipulating the probe to bring the sensing surface into contact with atear-bearing portion of a patient's eye. Moreover, providing the surfaceplasmon resonance apparatus with the sensing surface may comprisedisposing a metallic film carrying sheath in a predetermined position onan operative tip of the probe.

Pursuant to an alternative method of the present invention, the placingof the tear sample comprises extracting the tear sample from a patient'seye and depositing the tear sample on the sensing surface. The tearsample may be extracted and deposited using a micropipette.Alternatively, the tear sample may be extracted and deposited using anabsorbent porous material such as blotter paper.

Where the surface plasmon resonance apparatus includes a light-sensingdevice and a computer or microprocessor operatively linked to thelight-sensing device, the operating of the surface plasmon resonanceapparatus includes operating the light-sensing device to transmit to thecomputer an electrical signal encoding a pattern of light absorption bythe sensing surface and operating the computer or microprocessor toanalyze the image from the light-sensing device.

The operating of the computer or microprocessor preferably includesoperating the computer or microprocessor to compare an absorption-lineposition with prerecorded data correlating absorption-line position withosmolarity.

A medical diagnostic system comprises, in accordance with the presentinvention, a surface plasmon resonance apparatus having a sensingsurface for contacting a tear sample from an eye of a patient andfurther having a light-sensing device and a computer or microprocessoroperatively linked to the light-sensing device for receiving therefroman electrical signal encoding a pattern of light absorption by thesensing surface. The computer or microprocessor is programmed to analyzedata from the light-sensing device to determine a parameter related toosmolarity of the tear sample in contact with the sensing surface.

The light-sensing device may be a camera such as a charge-coupleddevice. In that case, the electrical signal is a video signal.Alternatively, the light sensing device may be a multi-elementphotodiode and the electrical signal. Then the electrical signal is ananalog difference signal.

The computer or microprocessor preferably includes means for comparingan absorption-line position with prerecorded data correlatingabsorption-line position with osmolarity.

In one embodiment of the present invention, the plasmon surfaceresonance apparatus includes a handheld portable casing with anoperative tip adapted to facilitate contact of a sensing surface on theoperative tip with a tear-bearing portion of a patient's eye.

The sensing surface of a plasmon surface resonance apparatus inaccordance with the present invention may include a first layer of afirst metal such as gold disposed on a second layer of a second metalsuch as chromium. Of course, the combinations or signal layers of metalsmay prove useful.

A feature of the present invention is its ability to measure nanolitervolumes of tears, a typical volume associated with normal and dry eyepatients. The apparatus is useful in determining the presence and degreeof dry eye syndrome, as osmolarity levels of tears are known tocorrelate with the severity of dry eye. The measurement of osmolarity isobtained virtually instantaneously once the sample is placed on thesensing surface, and therefore no problems of evaporation areencountered which could introduce errors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an optical schematic in cross section showing the basiccomponents of an osmometer in accordance with the present invention.

FIG. 2 a is a diagram of an image seen by a CCD camera of the osmometerof FIG. 1 when no sample is on a sensing surface.

FIG. 2 b is a diagram of an image seen by a CCD camera of the osmometerof FIG. 1 when a sample with a particular salt concentration is on thesensing surface.

FIG. 2 c is a diagram of an image seen by a CCD camera of the osmometerof FIG. 1 when a sample with an increased salt concentration is on thesensing surface.

FIG. 3 a is a graph showing the characteristic intensity versus anglecurve position where surface plasmon resonance occurs when a sample isdeposited on the sensing surface of the osmometer of FIG. 1.

FIG. 3 b is a graph showing the result from two different samples placedon the sensing surface of the osmometer of FIG. 1 and the resultingshift in the characteristic curve.

FIG. 4 a is a more detailed optical schematic of an osmometer inaccordance with the present invention.

FIG. 4 b is an optical diagram showing a more detailed view of thesensing surface, hemicylindrical prism, sample solution, and ray path.

FIG. 4 c is a diagram depicting the image on the CCD camera when thebeam is allowed to diverge in order to produce a broader line.

FIG. 5 is a schematic perspective view of a patient's eye and amicropipette showing the process of extracting a tear from the patientwith the micropipette.

FIG. 6 a is a schematic top view of the sensing surface of the osmometerof FIG. 4 a with a drop of solution covering the entire laser lineimage.

FIG. 6 b is a representation of the corresponding image seen by the CCDcamera.

FIG. 6 c is a schematic top view of the sensing surface of the osmometerof FIG. 4 a, similar to FIG. 6 a, showing a drop of solution coveringonly half of the laser line image.

FIG. 6 d is a representation of the corresponding image seen by the CCDcamera.

FIG. 7 a is a schematic top view of the sensing surface of the osmometerof FIG. 4 a with a nanoliter drop covering the small portion of thelaser line image.

FIG. 7 b is a representation of the corresponding image seen by the CCDcamera.

FIG. 7 c is a schematic top view of the sensing surface of the osmometerof FIG. 4 a, similar to FIG. 7 a, showing four nanoliter drops coveringseveral points along the laser line image.

FIG. 7 d is a representation of the corresponding image seen by the CCDcamera.

FIG. 8 a is a graph depicting the result of placing two differentsamples on the sensing surface. It shows the intensity of lightdiminishing and then rising again representing the dark line falling onthe CCD. The shift in the two curves represents a change in osmolarityand also shows the noise component present in the prototype system.

FIG. 8 b is a graph showing a straight line fit method of determiningthe shift between the two curves.

FIGS. 9 a and 9 b are representations of images generated using theosmometer of FIG. 4 a, depicting a data acquisition step.

FIG. 9 c is a graph depicting analysis and processing performed by acomputer or microprocessor of an SPR signal to determine osmolarity of atear.

FIG. 10 a is a schematic front elevational view of a quadrant photodiodedetector utilizable in an osmometer in accordance with the presentinvention.

FIG. 10 b is a view similar to FIG. 10 a but on a larger scale, showingtwo SPR signals superimposed on each half of the quadrant.

FIG. 11 is a schematic perspective view of a handheld SPR tear osmometerin the form of a hand-held probe, miniaturizing and simplifying theinstrument.

FIG. 12 is a schematic partial side elevational view of the hand-heldSPR tear osmometer probe of FIG. 11, touching the tear film of the eyein the area of the sclera.

FIG. 13 is a schematic exploded view of the SPR tear osmometer probe ofFIGS. 11 and 12, showing a disposable sensing surface and sheath,protective shell, optical core, and component holding back shell.

FIG. 14 shows the detailed optical schematic and ray path in and out ofthe optical core SPR Tear osmometer probe.

FIG. 15 is a schematic perspective view of a precision micropipetteholder that accurately positions a micropipette over the sensing surfaceof an osmometer in accordance with the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The invention will now be described in more detail by way of examplewith reference to the embodiments shown in accompanying figures. Itshould be kept in mind that the following described embodiments arepresented by way of example only and should not be construed as limitingthe inventive concept to any particular physical configuration.

A preferred embodiment of the present invention is shown in crosssection in FIG. 1. In SPR spectroscopy, we are interested at what angledoes the incidence light striking the sensing surface diminishes to zeroupon reflection. Light from a laser is focused by a cylindrical lens 102after reflecting from an angled mirror 104 into a line image 106 ontogold-coated sensing surface 108. Since the laser beam is convergent 110,multiple angles of light are incident on the sensing surface at once.The reflected light 112 is then collimated by a second cylinder lens 114after reflection from an oppositely angled mirror 116 to the first,falls on a charge-coupled device (CCD) camera 118. With no sample on thesensing surface, the resulting image seen by the camera is simply abright field 202, that is, all pixels on the CCD see the same amount oflight as shown in FIG. 2 a.

A main object of the invention is to determine the osmolarity of anunknown sample of solution whose volumes are on the order of nanoliters.Osmolarity is essentially the concentration of salt in water and can beexpressed in units of milliosmos (mOs). The following descriptionoutlines how the apparatus detects changes in osmolarity, with a moredetailed description to follow.

In the particular setup shown in FIG. 1, if a droplet of water isintroduced on the sensing surface 108, surface plasmon resonance occurs,and light from one of the angles in the convergent beam 110 iscompletely absorbed into the gold layer of the sensing surface. Theresulting image seen by the camera 118 is the same bright field 202 witha dark horizontal line 204 in the image as shown in FIG. 2 b. Now, if asalt crystal (i.e. table salt) is put into the droplet of water on thesensing surface, the dark line in the image 206 will shift upwardamongst the bright field 202 as the salt crystal dissolves (FIG. 2 c)essentially indicating a change in refractive index. This corresponds toa shift in the angle (delta θ) of light where the surface plasmonresonance occurs—i.e. a different ray in the convergent beam 110. If onewere to plot the intensity in the vertical (y) direction across theimage shown in FIG. 2 b, the characteristic SPR curve 302 will beobtained which is well defined by mathematical equations and is shown inFIG. 3 a. Increasing the salt concentration in the water drop on thesensing surface by adding additional salt will force the dark line tomove even further vertically. If one were to plot the intensity of thedark line in FIGS. 2 b and 2 c, a lateral shift in the SPR curve 304 isseen indicative of a change in the angle of where the phenomenon occurs,as shown in FIG. 3 b as a delta θ (306) and is directly related to thechange in osmolarity. Therefore with calibration, we can determine theexact salt concentration or osmolarity, of an unknown solution.

Practical System Considerations

A more detailed examination of the present invention will now bedescribed. Referring to FIG. 4 a, an SPR based nanoliter tear osmometeruses a laser diode module (diode laser plus collimating and circlizingoptics) 402 as the light source with a wavelength equal to 670 nm,however any of the various types of laser sources can be used—gas, solidstate, semiconductor, etc. For SPR to occur, the laser light must bepolarized in a plane parallel to the sensing surface, and therefore apolarization filter 404 is inserted into the laser beam ensuring oneplane of polarization. SPR can be observed at virtually any wavelengthof light, but will be described here using 670 nm (‘red’) since theprototype was designed to function at that wavelength.

Following the polarizing filter, a spatial filter (consisting of a shortfocal length lens and a pinhole) 406 is used to rid the laser beam ofany noise. Unfiltered laser beams will contain spatial noise fromparticles in the air, dust on lenses, etc., and the result will be abeam with unwanted diffraction patterns which can add complexity toimage processing during data analysis. The lens and the pinhole in thespatial filter are chosen for the particular laser wavelength tooptimize filtering (i.e. 6 mm focal length lens and 10 micron diameterpinhole). Light exiting the spatial filter is diverging and iscollimated with a spherical lens 408. After collimation the light entersa cylindrical lens 410 and the beam is focused to a line image 412. Thesensing surface 414 where the tear sample is to be placed needs to behorizontal (in the simplest case)—if it were vertical the tear samplewould have a tendency to fall off. Therefore, an angled mirror 416intercepts the beam before reaching focus and diverts the line image toa horizontally placed sensing surface 414. The reason a cylindrical lensis used (instead of spherical lens) is to produce a focused line image.Given this line image, a sample of tear can be placed anywhere on theline and an SPR signal will be seen; additionally multiple samples canbe analyzed at once by placing them along the length of the line image.

The cylindrical lens generates a converging cylindrical wavefront and afocused line image at the sensing surface. For SPR to occur, the laserlight must be in a dielectric medium (i.e. glass) and then strike themetallic gold sensing surface. This is usually accomplished by means ofan equilateral prism with the gold deposited on the based of the prism.However, the flat faces of the prism will introduce refraction of everyray incident, and to simplify the mathematics, a cylindrical ‘prism’ 417is used. This prism is a half of a cylinder made out of optical glass.It can be conveniently fabricated by centerless grinding a rod of aparticular glass and then polishing the outer diameter. The rod can thenbe ground in half along the axis of the cylinder and the flat of thecylinder face is polished producing a ‘hemi-cylinder’. By using acylindrical prism, the surfaces introduce no refraction since all raysentering the prism are perpendicular to the surface. The sensing surfacecan simply be the polished face of the cylindrical prism and a thin filmof gold can be deposited directly on this surface. However, in practicethe cylindrical prism was made 1 mm less than a full hemi-cylinder andflat glass plates of the same material with the same dimensions as theflat face of the cylinder were made to 1 mm thick. It is one these glassplates where the gold is deposited making up the sensing surface, sinceit is simpler to fabricate many of these plates than prisms. Thecylindrical prism and glass plate with deposited gold are assembledtogether with index matching fluid, so to the incoming light theassembly appears as one unit (i.e. there are no changes in media andhence reflections between the cylinder prism and the glass plates). FIG.4 b shows a cylindrical prism 430 and a gold deposited sensing surface432 assembled with index matching fluid with a sample drop 433 on thesensing surface. Additionally is shows multiple angle light raysentering the prism at 434 and exiting the prism at 436, in each casewithout refraction.

Deposition of the gold onto the glass plates is a difficult process,since an exact thickness of gold is required for a strong SPR signal.Adding to the complexity, gold when vacuum deposited, does not stickvery well to glass, and as such simple cleaning of the surface wouldwipe away the deposited gold. To make the gold more robust, thewell-known technique of first depositing chromium is used. In oursystem, 2-5 nanometers of chrome is deposited followed by 50 nanometersof gold. It is somewhat difficult in practice to obtain exactly 50nanometers of gold, and several test runs were performed in order tocalibrate the evaporation coating chambers to achieve a useablethickness somewhere between 50 and 55 nanometers of gold. It turns outthe amount of chrome used can be critical as well. If too little chromeis used, the gold is not very durable and has a tendency to lift offduring handling or use. If too much chrome is used a low SPR signal isseen or even none at all.

Since the wavefront entering the prism is convergent, this means that arange of angles will be incident onto the sensing surface. The SPRminimum occurs at only one particular angle for a given solution. Forexample, if the media surrounding the sensing surface is air, the anglewhere SPR will occur (reflected intensity minimum) will be ˜33 degrees.If water is introduced as the media on the sensing surface in the formof a drop 433 as shown in FIG. 4 b, SPR will occur at 54 degrees. Sincethe object of the invention is to measure varying concentrations of saltdissolved in water, and there is a range of human tear osmolarities thatdetermine the presence and degree of dry eye, the apparatus has a rangeof angles (‘dynamic range’) that are within the range of SPR occurrencefor these solutions. For example, our system has a convergent beam of+/−5 degrees. The range of osmolarity of human tear is roughly 300 mOsto 400 mOs. In an ideal system we would be able to assign a fraction ofa degree to say 1 mOs, and determine osmolarity with extreme accuracy.In practice however, the change in angle as osmolarity changes is verysmall, on the order of 0.1 degree over the range of 300-400 mOs, whichintroduces challenges in signal detection.

Again referring to FIG. 4 a, after the light is allowed to focus to aline image it reflects and diverges from the gold sensing surface. If asample is in place, the reflected image exhibits a dark line, generallyshown in FIG. 3 a or b. This exiting light is reflected by a similarlyplaced mirror 418 at an opposite angle to the first, to bend the lightback onto the original optical axis of the laser. A second cylinder lens420 then collimates this diverging light. A charge-coupled device (CCD)camera 424 can be placed perpendicular to the light beam and a brightfield with a dark line is obtained as a video signal for laterprocessing. However, since our change in angle is small, on the order of0.1 degree, this translates to a vertical shift of the dark line fromthe extreme values of osmolarity to merely 10 or so microns, about thesize of a pixel in the CCD camera. Though it is possible to detectmovement of the line to less than a pixel by image processingtechniques, movement of the line can be increased optically. Instead, ashort focal length lens 422 can be inserted after the cylinder lens 420into the collimated beam that focuses the beam to a small point and thenallowed to diverge. If the CCD camera 424 is then placed after the beamis allowed to diverge, the beam quickly gets large, and the result is alarge dark line 438 (covering perhaps ¾ of the active area of the CCD442) surrounded by a bright field 440, shown in FIG. 4 c. In this case,the dark line covers many pixels as does its movement, and smallmovements can be more easily detected. The image on the CCD is capturedwith frame grabber 428 mounted in Personal Computer 426.

Measuring Tear Osmolarity Using SPR Spectroscopy

Now that the system intricacies have been adequately described, thefollowing will describe its use as a nanoliter tear osmometer.

There are inherent difficulties in handling nanoliter volumes ofsamples—the maximum amount of tear fluid usually able to be collectedfrom a patient. Extraction of the sample from the patient is relativelysimple, however inserting this sample into an instrument can causedifficulties. In osmometers using freezing-point depression technique,the sample must be frozen allowed to thaw, and the point of completethawing is related to osmolarity. Concern of evaporation comes into playwhen samples are small and if it occurs, an incorrect reading of theosmolarity will be obtained. Additionally the nanoliter tear sample mustbe put into some sort of freezing mechanism (i.e. thermoelectric coolingplate), which is observed microscopically. In short, it has been foundto be a very difficult process for freezing-point depression osmometersto obtain reliable data, or a device that is simple to use.Freezing-point depression based osmometers are much better suited atmeasuring larger (milliliter) size samples.

The interesting fact about SPR is that the phenomenon occursirrespective of the sample size. As long as the solution is in contactwith the sensing surface, and covers the focused (line) laser light, SPRwill occur. In the prototype apparatus, the width of the focused laserline was approximately 5 microns, and experimentation has shown thatnanoliter sample volumes produce identical SPR signals as millilitersize samples.

Calibration

To measure a tear sample osmolarity, the apparatus is first calibratedby way of laboratory made solutions of known osmolarity. Accuratelymeasuring Sodium Chloride and dissolving in triple distilled water madeseveral solutions with different osmolarity. Each one of these solutionswas deposited onto the sensing surface and a dark line forms in theimage at the same instant. The signal from the CCD camera which capturesthe image, is fed into a frame grabber 428 installed in a personalcomputer 426 and the image is digitized by the frame grabber and storedin the computer (FIG. 4 a). This process is performed for many solutionsand an image for each is obtained.

Now that the computer has several images of a bright field with a darkline, it detects the vertical movement of the dark line from solution tosolution. The result is a calibration curve that is stored in thecomputer for later recall. If now a solution with unknown osmolarity isdeposited onto the sensing surface, the computer can find the verticalposition of the line, compare it to the calibration solution data, andoutput the osmolarity of the unknown solution in units of milliosmos.

Flow Cell

To make the process of calibration—the deposition of many solutions withdifferent osmolarity on the sensing surface—simpler, a pump system and aflow cell was constructed. This system consists essentially of a fluidinput port, a small holding chamber, and a fluid output port. The unitrests atop the sensing surface and a silicone o-ring makes contact withthe sensing surface. The unit is screwed place and compression of theo-ring on top of the sensing surface creates a leak-proof seal. Attachedto the input port is tubing, the other end attached to a small pump. Theinput of the pump draws in the calibration fluid, sends it into the flowcell and passes through the holding chamber and out through the exitport. Once the fluid lands on the sensing surface a dark line appears inthe image at some vertical height, and it is recorded by the computer.To measure other solutions, the tubing leading to input of the pump isfirst inserted into water, to wash the sensing surface, followed by asolution of different osmolarity to the first, and the process isrepeated. This gives the computer a full set of data in a short periodof time if compared with dropping by hand one solution, recording theimage, removing the drop, washing with water, applying the next drop,etc.

Tear Extraction

The extraction of tears from a patient is performed with the use of aglass micropipette. Under slit-lamp observation, the physician placesthe micropipette 502 near the lower lid and just touching the tear strip504 so that tear fluid (˜200 nanolilters) capilates 506 into themicropipette (see FIG. 5).

The tip of the micropipette is tapered from the 3 mm capillary body downto about 0.5 mm, and is bent at an angle to the body. This is such thatduring tear extraction the body of the pipette can be angled away fromthe face and that the process does not stimulate excess tear productionwhich can skew the osmolarity reading.

Now that the tear sample is contained in the micropipette tip, there issome difficulty in getting the tear out and placed exactly. This isroutinely done by hand, however in a commercial instrument a moreconvenient manner is needed. Air pressure must be introduced to overcomethe capilating action to force the tear out of the micropipette. Thiscan be done with a squeeze bulb or by simply blowing on the end. Thedominant issue with handling nanoliter sample volumes is that they areso small and all forces (i.e. surface tension) near the sample aregreater than the mass of the sample, which consequently has a tendencyto be pulled to whatever object is closest to it or has the greatestsurface tension. When the tear is forced out of the micropipette withgentle air pressure, the tear sample will have a tendency to exit themicropipette and roll up the side of the pipette due to surface tension.However, the tear sample if upon exiting the pipette yet still attachedto the end is put in close proximity of another surface (i.e. thesensing surface) the surface tension from the sensing surface pulls thesample onto the surface. This takes a considerable amount of skill andpractice. Further complicating matters, the tear sample must liedirectly on the focused laser beam on the sensing surface. Therefore, asystem is contemplated that pulls the tear sample out of themicropipette and accurately places it on the sensing surface. One methodis the use of a microdispensing system that is commercially available.This system uses precise air pressure to force an exact amount of fluidout of a syringe (in this case a micropipette) and this in combinationwith a micropostioning system that will bring the micropipette tip inclose proximity with the sensing surface and will allow for accuratedispensing and placement of the tear sample on the focused laser lineimage. Other work has been done in the area of using electrical force todispense a small droplet of fluid.

Placement of the tear on the sensing surface can be simply done with a3-axis (translation) micropositioning system, which holds themicropipette vertically with the tip facing the sensing surface.Adjustment of the 3 axes via control screws until the tip is right overand nearly in contact with the sensing surface can be done by hand andvisual inspection. However, this is inconvenient (and potentiallyexpensive) in a commercial product. The manufacturing of custommicropipettes to a specific tolerance such that the micropipette can beinserted into a fixture mounted over the sensing surface and itsposition be repeatable is one method for simple accurate positioning ofthe tear on the sensing surface. FIG. 15 shows a fixture 1502 that wasused to position up to three micropipette tips 1504 over and along thefocused laser line 1506 on the sensing surface 1508.

One other method for extracting the tear can be done with absorbentmaterial (i.e. paper) that when touched to the tear film or stripabsorbs the tear sample. This wet paper then can be touched to thesensing surface and a dark line will appear in the image as before. Theadvantage to using a paper strip to absorb the tear sample is that aslit lamp microscope is not needed for extraction.

Image Characteristics and Sample Size

Depending on how a solution under test is deposited onto the sensingsurface determines the characteristics of the final image. However, allmethods will reveal a dark vertical line, surrounded by a bright field.Referring to FIG. 6 a, the top view of the rectangular sensing surfaceis shown at 602. The cylindrical lens in the system focuses laser lightto a line image 604 on the sensing surface as shown in the figure. Onthis sensing surface and covering the line image the sample solution 606is deposited. The corresponding image is a horizontal dark line 608 in abright field 610 that translates vertically with change in osmolarity.The length of this line however is determined by the amount of thesolution that covers the focused laser line. If the solution 612 onlycovers half the laser line 614 on the sensing surface 616 as shown inFIG. 6 b, the resulting dark line 618 in the image will only cover halfof the CCD camera, and the rest will be a bright field 620.

However, when dealing with tears of nanoliter volumes 702, when placedon the sensing surface 704, will only cover a small fraction of thelaser line image 706. The result in the image on the CCD will be abright field 708 with a ‘short’ dark line 710. This is shown pictoriallyin FIG. 7 a. As the osmolarity of the tear changes, this short line willmove vertically in the image. Note that since the sensing surface islarge compared to the volume of the tear, it is conceivable that morethan one tear (i.e. right eye and left eye) can be placed on the sensingsurface at one time, giving immediate readings of both. Additionally,several tears can be placed on the surface at once getting multiplereadings. FIG. 7 b shows four tear samples 711, 712, 714, 716 withdifferent osmolarity on the sensing surface 718 and the resulting imageon the CCD camera showing four dark lines 722, 724, 726, 728 atdifferent vertical positions amongst a bright field 730. The differentvertical positions of the lines indicate different osmolarity of eachsample.

Image Process Routines for Determination of Vertical Position of DarkLine

As described earlier when a solution is introduced onto the sensingsurface the image seen by the CCD camera is shown in FIGS. 2 a and b. Inpractice however, this image does just contain a bright field and a darkline, but also noise (bright and dark patches) from spurious reflectionsand unwanted diffraction from many sources, even with a spatial filterin place. Noise in general can cause an uncertainty in determining theposition of the dark line. However, even with this noise, thecharacteristic SPR signal of diminishing intensity (see FIG. 3) can beseen and with simple averaging a reliable measurement can be made.However, when measuring osmolarity, differences need to be detected onthe order of 1 mOs or less, representing a very small change in SPRangle or equally the vertical position of the dark line in the image.Therefore is necessary for the computer to process each image capturedfrom the CCD camera to remove the noise component, or at least reduceit.

The image of the dark line shown in FIG. 2 is not representative of theactual image obtained with the system. In an ideal system with an SPRsignal present, if the intensity of the light falling on the CCD camerawere plotted vertically along one column of pixels of the CCD the resultwould be the curve shown in FIG. 3. In reality, this curve has riding onit random noise components. To precisely determine the osmolarity of asolution, the computer must find the minimum of this curve, and compareit to the calibration tables. The more accurately the computer canautomatically determine the location of the minimum, the more accuratethe measurement of osmolarity will be. With too much noise present, thecomputer can mistakenly interpret noise as a SPR minimum and report thewrong value of osmolarity. Additionally with noise present, the computermay not be able to detect a small change in movement of the verticalline when solutions close in osmolarity values are measured (i.e. 312.0mOs and 312.5 mOs). For osmolarity measurements to be useful clinicallythe system preferably reports osmolarity to within 1-2 mOs of it actualvalue. Theoretically the system would be able to detect changes down to0.1 mOs which is likely to be insignificant from a clinical standpoint.

With no sample on the sensing surface the ‘baseline’ image consists of abright patch of light. In this bright patch of light contains (spatial)noise. This noise generally consists of diffraction patterns and otherpatches from interference caused by small scratches or pits in the lens,dust particles, etc. Additionally, the intensity of laser light isGaussian distributed. All of these unwanted features in the baselineimage as present when a sample is placed on the sensing surface and adark line forms due to SPR. To remove this unwanted spatial noise, thecomputer can subtract the two images thereby removing these features inthe processed image. However, there occurs an amplitude differencebetween the baseline image and the image with the dark line, so ascaling factor is determined by analyzing the same small portion of eachimage and comparing the average pixel value between the two areas. Theresult is a dark field with a bright white line, with the spatial noisecomponent minimized.

There are other sources of noise still present in the signal even aftersubtraction. These are predominantly random fluctuations such aselectronic noise leading to difficulty in determining the exact location(or change in location) of the dark line.

One method is to not only monitor the pixel of minimum intensityrepresentative of the minimum of the dark line, but to monitor it andseveral pixels around it. The lower portion of the SPR curve containingthe point of minimum intensity can be approximated as a parabola. Thecomputer program fits a parabola to the points surrounding the minimum,and then monitors all points on the parabola. In this manner, if theparabola shifts even less than one pixel, the computer can detect it,generally known as subpixel resolution.

If the line is broadened using the diverging lens shown in FIG. 4 a itachieves a larger vertical movement of the dark line. If one were toplot the intensity from top to bottom of the CCD, a truncated version ofthe curve in FIG. 3 a will result. If two solutions are tested and theintensity on the CCD is examined at each instant, two broadenedtruncated curves will be obtained. Actual curves from two solutionstested on the prototype instrument are shown in FIG. 8 a. These curvesshow the diminishing intensity of the laser light reflected from thesensing surface with a sample in place, and contain noise, seen in thefigure as high frequency ripples in an otherwise smooth curve. Eachcurve is shifted slightly to each other indicating a change inosmolarity. The distance between these curves or the shift is measuredto determine the value of osmolarity for given solution.

One way to measure the shift between the curves shown in FIG. 8 a, is tofit a straight line to each side of the curve. The point where theselines cross can be noted, and this performed for each curve. This willyield two distinct points separated by a distance, and this distance isrepresentative of the shift in the actual curves. This is shown in FIG.8 b.

Another method of successful detection of the vertical position of thedark line was to find the average position, plot the data and find theweighted center of the curve. It is beneficial to normalize the datafirst so that cut off values will not need to be determined for eachsolution. FIG. 9 shows the process of data analysis using the centroidmethod. Placement of a tear sample upon the sensing surface generates animage on the CCD with a dark line covering a portion of the horizontalfield amongst a bright field at some vertical position 902. A frame ofvideo is acquired and stored on a PC. A computer program may be writtento allow the user the ability to select a region of interest 904 in theimage namely the dark line and a portion of the bright field above andbelow the line with a click and drag of a mouse. A button is pressed andthe software begins averaging the selected region of interest selectedwith the mouse. The software averages the brightness of all the pixelsin each horizontal row of the region to reach one brightness value foreach row. The resulting data can be thought of as the region collapsedinto a single vertical line, of the same height as the original region,where each point on the line is as bright or dark as the average of thewhole sideways row of the original region at that height. This producesa single curve representing the average vertical position of the darkline 906.

Adding a sample of different osmolarity causes a shift of the curve,producing two similar shaped but laterally translated SPR curves 906 and908. The processing continues to create a cutoff level 910 to separatethe peak from the rest of the curve, and calculates thecenter-of-gravity or centroid of the remaining data points in the peak.This method is more robust than choosing the single apex of the curve,since it involves more data near the curve in its final result, and somecurves may be oddly asymmetrical, and give inaccurate apexes. Each ofthe curves peak sections generated from solutions of differentosmolarity are centroided and a tick mark 912 is drawn below andassigned a value on a horizontal scale, which can be calibrated to theosmolarity value of the solution.

Photodiode Method and Probe Design

Another configuration of an SPR tear osmometer involves the replacementof the CCD camera with a multi-element photodiode. Miniaturization isconceived to play a role in the success of a commercial medical deviceas practical reasons prevent the practicing physician from using everytechnological instrument that is available in their field, simply due tolack of office space. To remove the CCD camera from the system may notsave substantial money from the component standpoint; it will save inpost processing hardware (i.e. frame grabber, computer). The use of aCCD camera merely simplifies things for the research staff, and use of acomputer allows one to make changes quickly. Computers are problematicand add expense so it is desirable from many fronts to have standalone,even portable devices.

An SPR tear osmometer system was built with all the same components asdescribed above, yet with the CCD camera (FIGS. 1 and 4) being replacedwith a 4-element (quadrant) photodiode (UDT Sensors, Inc. SegmentedQuadrant Photodiode). Shown in FIG. 10 a, a “quad detector” isessentially 4 photodiodes 1002 on the same substrate with a smalldeadspace 1004 in between. In this case the vertical position of thedark line is determined by allowing a portion of the dark line to beabove and a portion below the upper and lower quadrants as shown in FIG.10 b. The right upper and lower quadrants 1006 might represent a sampletaken from the right eye with a particular osmolarity value and verticalposition of the dark line 1008, and the left upper and lower quadrants1010 a sample from the left eye with corresponding dark line 1012. Aphotodiode generates a current when light is incident on the surface.The upper and lower photodiode will each generate their own currentbased the amount of light incident upon them which is affected by theposition of the dark line. In practice the currents are amplified(Burr-Brown Corp. OPA-128 Current Op-Amp) and converted to voltageswhich can be compared and the relative position of the dark line (SPRminimum) be inferred. Very similar processing can be done as describedwith the image obtained from the CCD, however in the photodiode case oneis dealing with analog voltages instead. Analog voltages can bedigitized with a computer or preferable a microprocessor which canoperate on the digitized voltages to produce a value corresponding tothe position of the dark line and hence the osmolarity of the sample.

The use of a photodiode as the detector and integrated electronics forsignal processing, micro-optics, and novel prism configurations candramatically reduce the size of the entire system, and would make for aproduction instrument that could be produced in high volume relativelyinexpensively. Though numerous configurations for SPR spectroscopy havebeen proposed for analysis of chemical and biological agents, none havebeen specific to tear osmolarity and the difficulties associated withnanoliter sample size, resolution, sample placement, etc.

The level of complexity of the optical system of an SPR spectroscope issimple enough to be miniaturized into a handheld probe. As long as theangle of incident light is correct, SPR will occur no matter what thevolume of the solution is—that is rather than probing the tear strip andcapilating tear fluid into a micropipette, the sensing surface of theinstrument can come in contact with the tear film itself. This cuts outa step of having to extract the tear, and solves the problems of tearplacement on the sensing surface. FIG. 11 shows a computer model of atear osmometer probe, and a handpiece 1102 of the probe includes abutton 1106 that is pressed to make the measurement, contains all thenecessary components of the SPR system.

The probe has a tip 1104 that is optical glass, small in diameter,approximately 3 mm by 0.5 mm thick. This tip 1104 is where the gold isdeposited on the optical glass and comes in contact with the tear film1202 anywhere on the eye 1204 (preferably the sclera) momentarily (FIG.12). Referring to FIG. 13, the angle of incidence necessary to measuretear osmolarity is maintained by two facets that act as first surfacemirrors 1302 and are part of the core 1304 of the handpiece 1102. Thecore is shown to be a solid piece of optical material with flats groundand polished for the angled mirrors 1302. Since the light is in thedense (glass) medium before striking the sensing surface no prism isneeded under this configuration simplifying and miniaturizing thesystem.

Since the metallic coating used in SPR is so thin, it is not verydurable and cannot stand up to repeated wiping. Also, sensing surfacescontaminate easily and degrade the SPR curve (add noise). And since thisinstrument is intended to be used on the general population, it isdesirable in measuring tear osmolarity to have a disposable sensingsurface which this design lends itself to. This is shown as a sheath1306 that holds the sensing surface disk 1308 and the entire system(sheath and disk) are disposed of after use.

The core of the system is housed in a mounting shell 1310 that protectsthe core and facilitates mounting of the sheath. The back shell 1312contains the rest of the components for the SPR system and also housesthe button 1106 to initiate measurement. Power and signal output areprovided via a cable 1314 exiting the back shell and signal is processedusing an external processing unit that contains a microprocessor (or acomputer).

The individual optical components that are housed in the back shell areshown in FIG. 14. Light from a laser diode 1402 is collimated by acollimating lens 1404 and passes though a polarizing filter 1406. Afocusing lens 1408 focuses light to a line 1410 onto a sensing surface1412 by reflecting off an integrated mirror 1414 formed by the coresurface. Light reflected from the sensing surface diverges and reflectedoff an integrated mirror 1416 and collimated by a collimating lens 1418.A multi-element photodiode 1420 detects the electrical SPR signalencoding a pattern of light absorption by the sensing surface. Acomputer or microprocessor is operated to analyze the image from thelight-sensing device, i.e., photodiode.

1. A medical diagnostic method comprising: providing a surface plasmonresonance apparatus; providing said surface plasmon resonance apparatuswith a sensing surface; placing a tear sample from an eye of a patientinto contact with said sensing surface; and operating the surfaceplasmon resonance apparatus to determine osmolarity of the tear sample.2. The method defined in claim 1 wherein said surface plasmon resonanceapparatus takes the form of a portable probe, the placing of the tearsample comprising manipulating the probe to bring said sensing surfaceinto contact with a tear-bearing portion of a patient's eye.
 3. Themethod defined in claim 2 wherein providing said surface plasmonresonance apparatus with said sensing surface comprises disposing ametallic film carrying sheath in a predetermined position on anoperative tip of the probe.
 4. The method defined in claim 1 whereinproviding said surface plasmon resonance apparatus with the sensingsurface comprises disposing a metallic film carrier in a predeterminedposition on the surface plasmon resonance apparatus.
 5. The methoddefined in claim 4 wherein the metallic film carrier is a plate, thedisposing of the metallic film carrier including placing said plate ontoa cylindrical prism of said surface plasmon resonance apparatus.
 6. Themethod defined in claim 1 wherein the placing of the tear samplecomprises extracting the tear sample from a patient's eye and depositingthe tear sample on said sensing surface.
 7. The method defined in claim6 wherein the extracting of said tear sample includes using amicropipette.
 8. The method defined in claim 6 wherein the extracting ofsad tear sample includes using an absorbent porous material.
 9. Themethod defined in claim 1 wherein the surface plasmon resonanceapparatus includes a light-sensing device and a computer ormicroprocessor operatively linked to said light-sensing device, theoperating of the surface plasmon resonance apparatus including:operating said light-sensing device to transmit to said computer anelectrical signal encoding a pattern of light absorption by said sensingsurface; and operating said computer or microprocessor to analyze theimage from the light-sensing device.
 10. The method defined in claim 9wherein said light-sensing device is a camera and said electrical signalis a video signal.
 11. The method defined in claim 9 wherein said lightsensing device is a multi-element photodiode and said electrical signalis an analog difference signal.
 12. The method defined in claim 9wherein the operating of said computer or microprocessor includesoperating said computer or microprocessor to compare an absorption-lineposition with prerecorded data correlating absorption-line position withosmolarity.
 13. A medical diagnostic system comprising a surface plasmonresonance apparatus having a sensing surface for contacting a tearsample from an eye of a patient and further having a light-sensingdevice and a computer or microprocessor operatively linked to saidlight-sensing device for receiving therefrom an electrical signalencoding a pattern of light absorption by said sensing surface, saidcomputer or microprocessor being programmed to analyze data from saidlight-sensing device to determine a parameter related to osmolarity ofthe tear sample in contact with said sensing surface.
 14. The systemdefined in claim 13 wherein said light-sensing device is a camera andsaid electrical signal is a video signal.
 15. The system defined inclaim 13 wherin said light sensing device is a multi-element photodiodeand said electrical signal is an analog difference signal.
 16. Thesystem defined in claim 13 wherein said computer or microprocessorincludes means for comparing an absorption-line position withprerecorded data correlating absorption-line position with osmolarity.17. The system defined in claim 13 wherein said plasmon surfaceresonance apparatus includes a handheld portable casing with anoperative tip adapted to facilitate contact of a sensing surface on saidoperative tip with a tear-bearing portion of a patient's eye.
 18. Thesystem defined in claim 13 wherein said sensing surface includes a firstlayer of a first metal disposed on a second layer of a second metal. 19.The system defined in claim 18 wherein said second layer is in turndisposed on a substrate of transparent material.